1. Field of the Invention
The present invention relates to high-throughput assays for characterizing a subject's genetic makeup. Specifically, the instant invention is a high-throughput assay that permits the rapid and accurate characterization of a subject's inherited alleles of the polymorphic glutathione S-transferase (GST) genes GSTM1, GSTM3, GSTP1, and GSTT1.
2. Background of the Invention
Recent cancer research has shown that the presence of various polymorphisms of glutathione S-transferase (“GST”) correlates with altered risk for certain cancers and altered response and toxicity from currently known and used cancer treatments, including chemotherapy. The GST family of enzymes has been shown to function in the detoxification of a broad range of environmental and non-environmental DNA-damaging carcinogens such as polyaromatic hydrocarbons like those found in first- and second-hand cigarette smoke. Additionally, however, these enzymes are capable of detoxifying chemotherapeutic compounds such as alkylating agents and nanthracyclines as well as reactive oxygen species and peroxides. See S. Tsuchida and K. Sato, Critical Reviews in Biochemistry and Molecular Biology, 27(4, 5):337–384 (1992), and S. A. Weitzman and L. I. Gordon, Blood, 76(4):655–663 (1990).
This family of enzymes has been subdivided into four subclasses, including GSTM1, GSTM3, GSTP1, and GSTT1. Ali-Osman et al., J. Biol. Chem., 272(15):10004–10012 (1997); Fryer et al., Biochem. J., 295:313–315 (1993); Inskip et al., Biochem. J., 312:713–716 (1995); and Pemble et al., Biochem. J., 300:271–276 (1995). A first of these is the GSTM1 class, which includes the following allelic variants: GSTM1*null, GSTM1*A, and GSTM1*B. Fryer et al., Biochem. J., 295:313–315 (1993).
GSTM1*null is thought to result from an unequal crossing-over at a duplicated region between the GSTM1 and the GSTM2 loci. Pearson et al., Am. J. Hum. Genet., 53:220–233 (1993); and Xu et al., J. Biol. Chem., 273:3517–3527 (1998). As with other null alleles, this one produces no functional product and thus acts as a recessive gene. GSTM1*A and GSTM1*B are polymorphic alleles commonly thought to result from a C to G substitution at codon 173. This change results in a change from the Lys173 of GSTM1*A to the Asn173 of GSTM1*B. This change alters a hinge region between alpha helices which is involved in GSTM1 dimerization.
A second subgroup of GST enzymes is dubbed the GSTM3 class, including the GSTM3*A and GSTM3*B allelic variants. These alleles are thought to result from a 3 base-pair deletion in intron 6 which generates a YY1 negative transcription factor recognition site in GSTM3*B that does not exist in GSTM3*A.
A third such subgroup of GST enzymes is the GSTP1 group which includes GSTP1*A, GSTP1*B, and GSTP1*C. A GSTP1*D allele has been observed, but only in very rare circumstances. These polymorphic alleles result from A to G and C to T transitions at nucleotides +313 of exon 5 and +341 of exon 6, respectively. Specifically, GSTP1*A codon 104 is ATC, coding for Ile104, and codon 113 is GCG, for Ala113. GSTP1*B codon 104 is GTC, coding for Val104, and codon 113 is GCG for Ala113. GSTP1*C codon 104 is GTC, coding for Val104, and codon 113 is GTG for Val113. GSTP1*D codon 104 is ATC, coding for Ile104, and codon 113 is GTG for Val113.
A fourth group of GST enzymes is the GSTT1 subgroup, which includes GSTT1*null and GSTT1. As with the other null allele noted above, the GSTT1*null allele produces no functional product, thus operating as a recessive allele.
There are techniques extant in the art for assessing which alleles are present in an individual's genotype. Most of these assays do not allow investigators to determine gene dosages. Here, the term “gene dosage” is used to denote whether one or both alleles were present when a PCR product suggested the presence of at least one non-null allele. Further, most of the currently used assays do not differentiate between the non-null GSTM1 alleles GSTM1*A and GSTM1*B. These methods also generally require that the PCR products undergo restriction endonuclease digestion to allow the determination of genotypes, thus adding extra complexity and expense to the method.
Kristensen et al. reported one such assay in 1998. Kristensen et al., Pharmacogenetics, 8:441447 (1998). This assay was able to evaluate only the polymorphisms of GSTM1, GSTP1, and GSTT1, while not being capable of distinguishing between GSTM1*A and GSTM1*B. Further, when looking at GSTP1, the assay examined only codon 104, ignoring the polymorphisms showing changes at codon 113. Finally, as with other known assays, this assay method did not examine the gene dosage of either GSTM1 or GSTT1.
The availability of simple, effective assays could allow the analysis of individuals' genomes in order to detect risk for specific diseases and cancers and to allow the development of individualized prevention and/or treatment strategies. Further, in regard to the specific family of enzymes referenced above, specific, accurate assays could allow the development of tailored therapeutic regimens for patients predicted to have decreased therapeutic response to medical therapy, including cancer therapy, based on their expression of GST enzymes, or for patients predicted to have increased therapy-related toxicity. Additionally, such assays would simplify the implementation of patient-specific utilization of allele-specific small-molecule inhibitors for the purpose of reversing chemotherapy resistance among cancers, such as those over-expressing certain GST polymorphic alleles.
From the above, it is apparent that it would be an improvement in the art to provide a high-throughput assay method for rapidly, inexpensively, and accurately characterizing the GST alleles present in a subject. It would be a further advancement in the art to provide a high-throughput GST assay method which is capable of accurately determining the gene dosage of GSTM1 and GSTT1 using competitive PCR. Additionally, it would be a further advancement in the art to provide such an assay design which is simpler, faster, and cheaper than those currently known in the art because it does not require restriction endonuclease digestion of PCR products in order to elucidate the length differences between GST alleles. Similarly, it would be an improvement in the art to allow the assay of all of the PCR products simultaneously in a single gel lane, which would yield further savings in time and expense. Finally, it would be an improvement in the art to provide a high-throughput assay method that would comprehensively assay all four GST polymorphs and their alleles, including null alleles. Such an assay method is disclosed herein.